Project Summary/Abstract To successfully colonize and establish themselves in the gastrointestinal (GI) tract, enteric pathogens must sense and respond to several microbiota and host derived signals to properly regulate expression of their virulence repertoire. An important signal within the GI tract is the host neurotransmitter norepinephrine (NE) and/or epinephrine (Epi), which have important functions in intestinal physiology. There is also an important relationship between neurotransmitters and the microbiota, because it modulates the levels of active Epi and NE in the gut lumen. The host conjugates Epi/NE to glucuronate to inactivate it. The microbiota encodes glucuronidases (encoded by the uidA gene) that deconjugate glucuronate from Epi/NE, increasing the levels of free biologically active Epi/NE in the lumen. We identified the first two bacterial adrenergic receptors, QseC and QseE. The enteric pathogen enterohemorrhagic E. coli (EHEC), exploits adrenergic signaling through QseC and QseE to carefully regulate expression of its virulence genes to promote optimal colonization of the gut. Importantly, EHEC virulence gene regulation intersects with the ability to sense glucuronate, linking deglucuronidation of Epi/NE by the microbiota to Epi/NE sensing at another level of inter- kingdom signaling. We identified ExuR, the sensor for glucoronate, as an important regulator of EHEC virulence gene expression. The linking of Epi/NE sensing through QseC and QseE, with glucuronate sensing through ExuR, ensures the precise control of virulence gene expression by EHEC. Indeed, Citrobacter rodentium (extensively used as a surrogate EHEC model for murine infections) qseC, qseE and exuR mutants are attenuated for murine infection, highlighting the important role of this signaling system in EHEC pathogenesis. Another neurotransmitter system, the endocannabinoid system is also intertwined with the gut microbiota. The endocannabinoid 2-Arachidonoylglycerol (2-AG) is sensed through QseC, preventing QseC function, and decreasing expression of virulence genes in EHEC and C. rodentium. Moreover, Epi and 2-AG antagonize each other at the level of QseC sensing. The levels of 2AG decrease from the small intestine to the colon, oppositely from the levels of Epi/NE. Given that EHEC colonizes the colon, the interplay between the adrenergic and endocannabinoid systems, likely has a key function in the biogeography of this pathogen within the GI tract. Accordingly the specific aims of this proposal are: Specific Aim 1: Investigate the relationship among the adrenergic and glucuronate signals in EHEC pathogenesis. Specific Aim 2: Investigate the role of the endocannabinoid system in EHEC pathogenesis. .